Molecular analysis of integrons and antimicrobial resistance profile in Shigella spp. isolated from acute pediatric diarrhea patients

Introduction: Shigella spp. is a growing global health concern due to increasing multiple drug resistance, commonly resulting in therapeutic failure. Integrons are gene expression systems run by integrase genes. The aims of this study were detection of class I, II and III integrons and assessment of antimicrobial resistance in Shigella spp. isolated from acute pediatric diarrhea patients. Materials and methods: From January to December 2015, 16 Shigella spp. were isolated from 310 non-duplicative diarrheal stool samples in Children’s Medical Center, Tehran, Iran. The isolates were analyzed for their antibiotic susceptibility using CLSI guidelines M100-S14. Multiplex PCR was used for amplification of I, II and III integron-associated integrase (intl) genes. Results: Of 310 stool samples, 16 (5.2%) were positive for Shigella spp., in 7 of them S. sonnei and in 9 of them S. flexneri were identified. Results of the antimicrobial susceptibility test showed that 6.2%, 50%, 31.2%, 6.2%, 81.2%, 56.2% and 31.2% of the isolates were resistant to gentamicin, chloramphenicol, nalidixic acid, ciprofloxacin, tetracycline, ampicillin and trimethoprim-sulfamethoxazole, respectively. Multiplex PCR results revealed that 6.2% (1/16), 31.2% (5/16), 50% (8/16) of Shigella isolates carried intlI, intlII and both intlI/intllI genes. No class 3 integrons were detected. Discussion: In this study, multidrug resistance was seen in Shigella isolates similar to that in isolates from other geographical areas. This is possible due to inappropriate use of antimicrobials. Furthermore, prevalence of multidrug resistance was significantly linked to the presence of integrin genes. Conclusion: A class 2 integron plays a role in presence of multidrug resistance in Shigella spp. It is vital to prevent the spread of antibiotic resistance through continuous monitoring.


Introduction
Dysentery caused by Shigella spp. is a major public concern worldwide and is responsible for approximately 5 to 10% of diarrheal diseases in many areas [1]. Recently in Asia, the number of dysentery cases was estimated at nearly 91 million, resulting in 414,000 deaths each year. In general, Shigella spp. are categorized into four serogroups, including S. dysenteriae, S. flexneri, S. boydii and S. sonnei [2]. Of these serogroups, S. flexneri is the most common, followed by S. sonnei. However, dysentery caused by Shigella spp. is usually self-limited, and antibiotic therapy is mostly effective not only in treating the dysenteric infection, but also in decreasing the duration of the disease and fecal shedding of the pathogen [3]. Over the last decades, Shigella spp. have increasingly acquired resistance to various antimicrobials, including ampicillin, streptomycin, tetracycline and trimethoprimsulfamethoxazole. The antibiotic resistance phenomenon in Shigella spp. commonly occurs due to mobile genetic elements (MGEs) such as R plasmids, transposons and integrons. Mobile genetic elements can mediate the distribution of resistance factors among the bacterial species, even genera. Furthermore, integrons with resistance gene cassettes have been recognized in MGEs. Resistance to antimicrobials in Shigella spp. is sometimes associated with class 1 and class 2 integrons, which comprise resistance gene cassettes. There are two types of class 1 integrons found in Shigella plasmids or chromosomes: atypical and classical integrons. These integrons are linked to gene cassettes of trimethoprim (dfrA1), esterase/lipase (estX), streptomycin (aadA1) and ampicillin (bla oxa30 ). Class 2 integrons carrying Tn7 are frequently present in S. sonnei and their gene cassettes contain dfrA1, streptothricin-acetyl-transferase gene (sat-1) and aadA1 [4], [5], [6], [7]. The aims of the current study were molecular analysis of integrons and antimicrobial resistance profiling in Shigella spp. isolated from acute pediat-ric diarrhea patients at the Children's Medical Center, Tehran, Iran.

Bacterial isolation
In a cross-sectional study, 310 non-duplicative and nonreiterative diarrheal stool samples were collected from children admitted to the Children's Medical Center in Tehran, Iran, from January to December 2015. Samples were transferred to the laboratory in Cary-Blair media (Merck, Germany). Samples were cultured, and the bacteria isolated and identified using conventional biochemical as well as microbiological methods in addition to the API-20E system (BioMerieux, France). Shigella polyvalent agglutinating antisera were purchased from MAST, UK.

Integron gene detection
Multiplex PCR (M-PCR) for detection of intlI, intlII and intlIII genes was carried out using a Master Cycler gradient PCR machine (Eppendorf, Germany). Microbial DNA was extracted using the boiling method from the colonies grown overnight on xylose lysine deoxycholate (XLD) agar. The primer sequences used in M-PCR are described in Table 1. The PCR reaction mixture was prepared in a total volume of 20 µl, consisting of 1 µl of template DNA, 2 µl of 10x PCR buffer, 0.6 µl of 50 mM MgCl 2 , 0.6 µl of 10 mM dNTPs, 0.5 µl of each primer, 0.7 µl of 5 U/µl Taq DNA polymerase (Amplicon, Denmark) and 12.1 µl of double-distilled water. The reaction mixture was transferred to a gradient thermal cycler (Eppendorf, Germany) with the following cycling program: initial denaturation at 94°C for 2 min followed by 33 cycles; each cycle included denaturation at 94°C for 30 s, annealing at 56°C for 30 s and elongation at 72°C for 30 s. Final elongation was carried out at 72°C for 10 min. Amplified products were visualized by electrophoresis in 1.5% agarose gels and staining with ethidium bromide.

Statistical analysis
Correlation between the occurrence of intlI, intlII and intlIII genes and multidrug resistance was calculated using Fisher's exact test. A P-value <0.05 was considered statistically significant.

Bacterial isolation
Of 310 stool samples, 16 (5.2%) samples were positive for Shigella spp. Of these 16 positive samples, 7 (43.7%) and 9 (56.3%) samples were identified as S. sonnei and S. flexneri, respectively. The mean age of the patients was six years, with 165 (53.2%) boys and 145 (46.7%) girls participating in the study. Nine (56.2%) bacterial species were isolated from children at ages 1 month to 2 years, and 7 (43.7%) in ages ranged from 2 to 12 years.

Statistical analysis
No significant difference was seen between the intlI gene and MDR (P>0.05). The correlation between the presence of intlII or intlI/II genes and antibiotic resistance was statistically significant (Table 4).

Discussion
Increased resistance of Shigella spp. to many antimicrobial agents presents a major threat to public health. Over the past decades, excessive use of antimicrobials and vast horizontal gene transfer have led Shigella spp. to become resistant to most routinely used antimicrobials. Primarily, tetracycline and sulfonamides were effective in the treatment of shigellosis, but the bacterial strains quickly established resistance to these agents. Later, ampicillin and trimethoprim-sulfamethoxazole were used to treat shigellosis. Antimicrobial resistance is common in Shigella spp., mostly to tetracycline, trimethoprimsulfamethoxazole and other sulfonamides. Increased bacterial resistance to ampicillin, chloramphenicol and trimethoprim-sulfamethoxazole is a serious threat. These are low-cost antimicrobials used widely for the treatment of shigellosis [8], [9], [10]. In general, multidrug-resistant Shigella spp. have been reported from Africa, Europe, Asia and South America. In the current study, all isolates were resistant to streptomycin. Thirteen (81.2%) and one (6.2%) Shigella isolates were resistant to tetracycline and gentamicin/ciprofloxacin, respectively. Similar results have been published from other studies in developing countries [11], [12], [13]. All isolates (100%) of S. sonnei were fully susceptible to gentamicin and ciprofloxacin. Moreover, 77.7% (n=7/9) and 11.1% (n=1/9) of S. sonnei isolates were resistant to tetracycline and trimethoprimsulfamethoxazole, respectively. The results showed that the highest and lowest resistance to ampicillin and gentamicin/ciprofloxacin in S. flexneri were 85.7% (n=6/7) and 14.3% (n=1/7), respectively. Pourakbari et al. reported that S. flexneri was more multiresistant than other species [14]. Results by Zhu et al. [15] showed that resistance of S. sonnei to ampicillin and ciprofloxacin varied and was relatively infrequent, while antimicrobial resistance was common in S. flexneri. Later, Shen et al. [16] demonstrated that resistance of S. flexneri (serotypes 1a) to antimicrobials was significantly higher, including 88.0%, 89.2%, 85.5% and 79.5%, to ampicillin, nalidixic acid, tetracycline and trimethoprim-sulfamethoxazole, respectively. In a similar study by Jafari et al. [17], most Shigella isolates were reported to be resistant to tetracycline (95%) and trimethoprim-sulfamethoxazole (91.7%). The maximum resistance (60.2%) was observed in S. sonnei. In the present study, multidrug resistance was detected in 57.1% (n=4/7) of S. flexneri and 33.3% (n=3/9) of S. sonnei isolates. This was in contrast to the results of the studies by Zhu et al. [15] and Jafari et al. [17]. This conflict may be due to geographical distribution, source of samples and level of hygiene.
Of the three classes of integrons linked to antimicrobial resistance, the class I integron is the most frequently found in clinical isolates of Gram-negative bacteria [18]. The class II integron is the most predominant integron in S. sonnei [5]. In the current study, 6.2% (n=1/16), 31.2% (n=5/16), 0% (n=0/16) and 50% (n=8/16) of Shigella isolates carried intlI, intlII, intlIII and both intlI/intlII genes, respectively. These results are similar to those of Shen et al. [16], Ranjbar et al. [19] and Nógrády et al. [20]. The present study has clearly shown that the prevalence of intlII is noticeably greater than that of intlI. Furthermore, the prevalence rate of these genes in MDR isolates with resistance to ≥3 drugs is higher than that in MDR [6], [15]. Zhu et al. [15] described that Shigella spp. included a high frequency of MDR and a high occurrence of classes I and II integrons at the same time; the prevalence of the intlII gene was significantly associated with MDR isolates (P<0.05) [21]. Currently, the presence of class II integrons and rate of MDR are linked in Shigella spp.; therefore, class II integrons may play a role in the presence of MDR in Shigella spp. This suggests a gene linkage between class II integrons and other antimicrobially resistant genes. Furthermore, this suggests that class II integrons work together with other determinants of genetic resistance. Further studies are needed to confirm these possibilities. The possible link of class II integrons with other antimicrobial resistance genes would help to employ class II integrons as molecular biomarkers to screen MDR in Shigella spp.

Conclusion
Antimicrobial resistance of Shigella spp. in developed countries appears to be frequent, and associated with their epidemiology in developing countries. Mostly, Shigella strains that carry class I or II integrons show emergence of MDR. Preventing the distribution of antibiotic resistance and spread of integrons is a matter of general urgency. Therefore, continuous monitoring schemes must be implemented to prevent further spread of MDR Shigella spp.

Competing interests
The authors declare that they have no competing interests.